The present invention relates to the measurement of the volume and hemoglobin concentration of individual red blood cells in a whole blood sample and, more particularly, to the making of such measurements for deriving therefrom statistical characteristics of the sample in automated clinical hematology instrumentation.
The red blood cells in a blood sample from a patient are not identical to one another. In each sample, there are statistical distributions of the volumes (V) and of hemoglobin concentrations (HC) of the individual cells. Consequently, the red blood cells in a sample are characterized by the mean cell volume (MCV), the mean cell hemoglobin concentration (MCHC), the width of the V distribution (RDW), and the width of the HC distribution (HDW). These statistical distributions and their associated mean values and widths are used to classify and diagnose anemias. For example, in iron deficiency anemia and in thalassemia MCV is low, MCHC is low, and RDW is high. Hereditary spherocytosis is characterized by an elevated MCHC. In sickle cell anemia the MCHC is normal but HDW is elevated, and the HC distribution is skewed toward high HC values. In hemoglobin SC disease HDW and MCHC are both elevated.
The red cell indices MCV and MCHC of a blood sample can be calculated from a manually measured value of the red cell count (RBC) in the sample, a photometrically measured value of the hemoglobin concentration in the sample as a whole (HGB), and a hematocrit value (HCT) representative of the percent by volume of packed red cells in the sample. These known manual measurement techniques do not provide V or HC distributions, nor the widths of such distributions.
Microscopic examination of a blood smear permits a qualitative evaluation of the distributions of cell size and cell color, the latter being indicative of the hemoglobin concentration of the cells. Various classifications of the observed characteristics of the blood smear are then made. For example, an abnormally wide distribution of sizes is called anisocytosis and abnormally pale cells are labeled hypochromic, as described, for example, in M. Wintrobe et al, "Clinical Hematology", Lea and Febiger, Philadelphia, 1981.
Another known technique for making quantitative measurements of the HC distribution (or cell density profile) is to subject the cells in a sample to centrifugation, during which the cells distribute themselves throughout a column containing a buoyant medium having a density gradient. The resulting cell distribution along the column is then evaluated by photometric techniques.
Automated flow-cytometric hematology instruments are currently used in almost all laboratories and hospitals. Examples of such instruments include the Coulter S+IV by Coulter Electronics, Inc. of Hialeah, Fla.; the H6000 and the H*1 by Technicon Instruments Corporation of Tarrytown, N.Y.; the Celldyne 3000 by Unipath Corporation of Mountain View, Calif.; the ELT-8 by Ortho Instruments Corporation of Westwood, Mass.; the Sysmex M-2000 by Toa Corporation of Japan, and the System 9000 by Sorono-Baker Diagnostics, Inc. of Allentown, Pa. With only one exception, the H*1, these instruments detect and measure only one signal per red blood cell, and this signal is interpreted as being representative of the volume of the cell. Except for the Technicon H*1, no cell-by-cell hemoglobin concentration determination is made by the aforementioned instruments. Thus, such instruments can be used to derive a volume distribution and associated MCV and RDW values, as well as measure RBC and HGB. The value of MCHC must then be calculated from RBC, HGB and MCV using the formula EQU MCHC=1000.times.[HGB/(RBCxMCV)]. (1)
Instruments which do not derive an HC distribution are incapable of determining HDW. The Coulter S+IV, the Celldyne 3000, the Sysmex M-2000 and the System 9000 all utilize the electrical resistance pulse sizing method, commonly referred to as the Coulter principle, for measuring the cell volume. The H6000 and the ELT-8 use a volume determination technique based on measurement of the light scattered by a cell into a single, selected angular interval. The H*1 determines both V and HC using measurements of the light scattered by a sphered red cell into two selected angular intervals.
In instruments based on electrical resistance pulse sizing, individual red cells entrained in an electrolytic fluid medium are made to pass through a relatively small aperture through which an electric current is flowing, as explained, for example, in "Electrical Resistance Pulse Sizing: Coulter Sizing" by V. Kachel in Flow Cytometry and Sorting, M. Melamed et al, Eds. Chap. 4, Wiley-Liss, New York, 1990. Because red blood cells are extremely poor conductors of electricity compared with the electrolytic fluid, each time a cell passes through the aperture, a change in electrical resistance of the electrolytic fluid within the aperture is measured by the instrument. The relative change in resistance, .DELTA.R/Ro, is related to the ratio of the volume of the blood cell to the volume of the aperture, V/Vo, through the equation EQU .DELTA.R/R.sub.o =f.sub.s (V/Vo), (2)
where R.sub.o is the resistance of the electrolytic fluid within the aperture when no red blood cell is being passed, .DELTA.R is the change in resistance when a cell is being passed through the aperture, V.sub.o is the volume of the aperture and V is the volume of the cell. The quantity f.sub.s depends on the shape of the red blood cell when it is in the aperture and is called the "shape factor".
The higher hydrodynamic forces acting on each cell as it passes through the aperture tend to deform the cell into a prolate spheroid. Theoretical considerations show that the shape factor, f.sub.s, varies between 1.0 for a very thin needle-shaped cell and 1.5 for a sphere. The amount of deformation experienced by a given red blood cell depends on its internal viscosity which, in turn, depends primarily on the hemoglobin concentration, HC, within the cell.
The value of HC is known to vary substantially among individual red blood cells in both normal, i.e., HC in the range of 27 to 40 g/dL (g/dL=grams/deciliter), and pathologic, i.e., HC in the range of 25 to 50 g/dL, blood samples. This wide variation in HC results in a correspondingly wide variation in the shape factor, f.sub.s, from cell to cell. In each of the above-mentioned instruments which use resistance pulse sizing, the assumption of a constant value for f.sub.s is made, and the empirical cell-to-cell variation in f.sub.s is ignored.
This expedient of assuming a constant f.sub.s follows from the fact that a single measurement of the resistance change .DELTA.R can only determine the product f.sub.s.V, as shown by equation (2). Therefore, by assuming a constant f.sub.s a value for V is easily obtained for each value of .DELTA.R. However, as a result of this assumption, the calculated MCV and the derived MCHC may be inaccurate, especially when the blood sample has a high proportion of cells with extremely low or extremely high hemoglobin concentrations. Cells with lower HCs (i.e., lower internal viscosities) tend to be more needle-like than cells with higher HCs (i.e., higher internal viscosities). Accordingly, the assumption of constant f.sub.s causes the volumes of low HC cells to be underestimated and the volumes of high HC cells to be overestimated Thus, from equation (1) blood samples having many red blood cells with elevated HC values will result in a measured value of MCV which is too high and a derived value of MCHC that is too low. Likewise, if the sample has many red cells with low HC values, the measured value of MCV will be too low and the derived value of MCHC will be too high.
These errors which result from the assumption of a constant f.sub.s in resistance pulse sizing measurements restrict the dynamic range of measured MCHC values relative to the true range of MCHC values. A serious consequence of this so-called MCHC/MCV interference is that present day instruments using resistance pulse sizing do not track variations in MCHC with sufficient accuracy. This introduces a systematic error which reduces the clinical value of MCHC as a red blood cell index, as explained, for example, in N. Mohandas et al, Blood 56, 125, 1980 and T. Arnfred et al: Scand. J. Clin. Lab. Invest. 41, 717, 1981.
In U.S. Pat. No. 4,298,836 to Groves and Coulter, a slit-scanning, time-of-flight optical technique for measuring the length of a cell is combined with resistance pulse detection in an attempt to determine the shape factor f.sub.s for each individual cell as it passes through the sizing aperture. If f.sub.s is known for each cell, an accurate volume could be derived from the resistance pulse signal. However, the length measurement of the cell is made after it leaves the sizing aperture, and owing to the substantial hydrodynamic forces on a cell as it exits a sizing aperture, it is unlikely that the measured cell length is the same as the length of the cell while in the aperture. Furthermore, this patent does not teach any technique for measuring the hemoglobin concentration of individual red blood cells.
An article by Bator et al., Cytometry 5, 34, (1984), describes a technique in which resistance pulse sizing is combined with an ultra high speed photographic technique to record on film images of red blood cells as they exit from the sizing aperture. Cell dimensions and f.sub.s are then determined manually from the photographs to allow corrections to be made to the resistance pulse volume measurements. The technique described in the Bator et al. article is much too slow to be applicable to present-day clinical laboratory instrumentation where typically more than 20,000 red cells per patient sample are counted and sized in 60 seconds or less. Furthermore, the Bator et al. article also does not disclose any technique for measuring the hemoglobin concentration of the individual red blood cells.
The electrical resistance pulse technique has been combined with optical measurements in a number of instances. U.S. Pat. No. 3,710,933 to Fulwyler et al. describes making optical measurements on biological cells after the cells have passed through a resistance pulse sizing aperture. The optical measurements are specified as being measurements of the intensity of forward scattered light within small angular intervals (i.e., angles between 0.5.degree. and 2.0.degree.) and measurements of the intensity of fluorescence emitted by the cells. These measurements do not provide the information required for determining the hemoglobin concentration of red blood cells or for correcting the accuracy of the resistance pulse volume measurements.
In an article by Thomas et al., "Combined Optical and Electronic Analysis of Cells with the AMAC Transducers", J. Histochem. Cytochem 25, 827 (1977), an automated multiparameter analyzer for cells is described in which optical measurements on a particle are carried out while the particle is within the resistance pulse sizing aperture. The only optical measurements described in the article are measurements of the intensity of fluorescence from small beads and leukemic white cells. Such measurements do not provide information for determining the hemoglobin concentration of red blood cells or for correcting resistance pulse volume measurements.
A similar apparatus is described in U.S. Pat. No. 4,198,160 to Kachel et al. The apparatus disclosed in this patent also combines fluorescence measurements with resistance pulse volume measurements. However, the fluorescence measurements, which are used to make determinations of other characteristics of the cells, do not provide the information needed to correct the accuracy of the resistance pulse volume measurements or to determine the hemoglobin concentration of individual red blood cells.
U.S. Pat No. 4,735,504 to the present inventor discloses a technique for determining the volume and hemoglobin concentration of individual red blood cells. The technique described in this patent uses two forward light scattering measurements made using different angular intervals. Because each of two measurements provides scattered light intensity signals which are non-linear functions of V and HC, this technique has the problem in that extracting the values of V and HC from the two measurements requires extensive computing resources, such as a computer with a high speed arithmetic unit and a large memory. Furthermore, this technique has the disadvantage of the higher complexity and cost associated with having two optical channels.
Accordingly, from the foregoing it is clear that a need exists for an improved apparatus and method for measuring the volume and hemoglobin concentration of individual red blood cells of a blood sample and for deriving useful characteristics of the sample, such as MCV, MCHC, RDW and HDW, without requiring extensive computing resources, and being otherwise less complex and of lower cost.